579
Inorg. Chem. 1990, 29, 579-581 [Fe111(2-BIM),]C1,,124756-06-5; [Fe'i(2-BIK),](CIOI)2, 124756-08-7; [ Fe1ii(2-BIK)3]3t,124756-09-8; 2-BIM, 64269-81-4; I-MeIm, 616-47-7. Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques Uniti Associee au CNRS en developpement concert& avec I'INSERM, No. 400 Universite Rene Descartes 45 rue des Saints-Ptres 75270 Paris Cedex 06, France
Table I. X-ray Absorption Spectroscopic Data Collection and Reduction sample(s) urease (native and 2-Me-treated) edges EXAFS SR facility SSRL SSRL beam line VII-3 11-2 (focused) monochromator crystal Si[400] Si[lll] detection method fluorescence fluorescence detector type Ar ion chambera 13-element solid-state arrayb scan length, min 17 24 av no. of scans 3 13-14 metal concn. mM 2. I 1.4 temp, K 9 11 energy standard Ni foil (1st inflcn) Ni foil (1st inflcn) energy calibration, eV 833 I .6 833 1.6 8350 8350 E,. eV preedge bkgd energy range, 8020-8300 (2) 8370-9000 (2)c eV (polynomial order) spline bkgd energy range, 8390-8732 (2) 8370-8531 ( 3 ) eV (polynomial order) 8531-8750 (3) 8750-9000 (3) " E X A H Co., Seattle, WA. *Courtesy of S. P. Cramer, National Synchrotron Light Source, Brookhaven National Lab~ratory.'~ 'The background was calculated from fitting this (EXAFS) region; then a constant was subtracted so that the background matched the data just bcforc the edge.
C.Cuillot E. Mulliez P.Leduc J.-C. Chottardl
Received March 2, 1989
X-ray Absorption Spectroscopic Evidence for Binding of the Competitive Inhibitor 2-Mercaptoethanol to the Nickel Sites of Jack Bean Urease. A New Ni-Ni Interaction in the Inhibited Enzyme Jack bean urease (EC 3.5.1S ) , the first nickel-containing metalloenzyme identified,l catalyzes the hydrolysis of urea to carbon dioxide and ammonia. The enzyme consists of a hexamer of identical subunits, each containing two nickel ions and one catalytic site.2 While the biochemical properties of urease have been characterized,' detailed physical studies of the nickel active site have been undertaken only recently. In particular, magnetic susceptibility measurements have now indicated a weak magnetic exchange interaction between the two paramagnetic Ni(I1) ions, providing evidence for a binuclear Ni(I1) active site in ~ r e a s e . ~ Further, competitive inhibitors have been shown to dramatically affect the ground-state electronic properties of the urease Ni(I1) ions. On addition of the competitive inhibitor 2-mercaptoethanol (2-ME; K , = 0.72 f 0.26 m M at 25 O C 3 ) , near-UV absorption bands arise that have been assigned as thiolate-Ni(I1) chargetransfer transitions, suggesting direct binding of the thiolate to the nickel ion(s) ( K d = 0.95 f 0.05 m M at 25 OC3). Binding of 2-ME to urease also causes the Ni(I1) ions to become diamagn e t i ~ Reported .~ herein are the results of a preliminary structural investigation using X-ray absorption spectroscopy (XAS) of the nickel sites of urease in its native and 2-ME-bound forms. This work confirms the direct binding of 2-ME to Ni(I1) through the thiolate sulfur. X A S has proven to be a useful structural probe of the active sites of nickel-containing the edge region yielding information about electronic structure (site symmetry, oxidation state, covalency)s and the extended X-ray absorption fine structure (EXAFS) region yielding the metrical details of the local nickel coordination environment. The urease Ni X A S data collection and reduction were accomplished as summarized in Table I.
I
I
I
!
8300
8320
I 1
I
I
I
8340
8360
I 1
1
8380 8400
Energy (eV) Figure 1. Comparison of the Ni K-edge X-ray absorption spectral region for (a) native urease (-) and [Ni(en),]CI2.2H20 ( - - - ) and (b) native (-) and 2-ME-treated urease (---). The insets show an expanded view of the region near the 8332-eV Is 3d transition.
( I ) Dixon, N. E.; Gazzola, C.; Blakeley, R. L.; Zerner, B. J . Am. Chem. SOC.1975, 97, 4131-4133. (2) Dixon, N . E.; Gazzola, C.; Watters, J. J.; Blakeley, R. L.; Zerner, B. J . Am. Chem. SOC.1975, 97, 4130-4131. (31 Blakelev. R. L.: Zerner. B. J . Mol. Catal. 1984. 23. 263-292. (4) Clark, P. A.; Wilcox, D. E. Inorg. Chem. 1989, 28, 1326-1333. (5) Scott, R. A.; Wallin, S. A.; Czechowski, M.; DerVartanian, D. V.; LeGall, J.; Peck, H. D., Jr.; Moura, 1. J . Am. Chem. SOC.1984, 106, 6864-6865. (6) Cramer. S. P.;Eidsness, M. K.; Pan, W.-H.; Morton, T. A,; Ragsdale, S. W.; DerVartanian, D. V.; Ljungdahl, L. G.; Scott, R. A. Inorg. Chem. 1987, 26, 2477-2479. (7) Eidsness, M. K.; Sullivan, R. J.; Schwartz, J. R.; Hartzell, P. L.; Wolfe, R. S.; Flank, A.-M.; Cramer, S. P.; Scott, R. A. J . Am. Chem. SOC. 1986, 108, 3120-3121. Shiemke, A. K.; Hamilton, C. L.; Scott, R. A. J . Eiol. Chem. 1988, 263, 561 1-5616. (8) Eidsness, M. K.; Sullivan, R. J.; Scott, R. A. in Eioinorgunic Chemistry of Nickel; Lancaster, J. R., Ed.; VCH: Deerfield Beach, FL, 1988; pp 73-91.
0020-1669/90/1329-0579$02.50/0 , ,
I
-
Urease was isolated, purified, and assayed as previously d e ~ c r i b e d . ~ 2-ME (15 m M ) was added by equilibrium dialysis. The native and 2-ME-bound samples had specific activities >74% and >68% (determined after removal of the thiolate inhibitor), respectively, of the maximum reportedg (2700 IU/mg), based on tzsO= 6.2 X IO4 M-I cm-I subunit-l; lower limits are reported because aggregation of jack bean urease results in increased absorbance at 280 nm due to light scattering and thus a lower specific activity based on protein concentration determined by AZs0.Comparison (9) Norris, R.; Brocklehurst, K. Eiochem. J . 1976, 159, 245-257.
0 1990 American Chemical Societv -
580 Inorganic Chemislry, Vol. 29, No. 4, 1990
-6 3.0
-6l
3.0
5.0
"
5.0
7.0 k
"
7.0 k
9.0
Communications
11.0
13.0
-$.O
1
-6l
'
510
(A-')
"
9.0
"
11.0
*
13.0
(A-')
3.0
"
5.0
'
710 k
"
7.0 k
'
9:O
'
1i.O
'
(A-')
"
9.0
"
11.0
'
Id.0
13.0
(A-')
Figure 2. Comparison of raw EXAFS data and analyses for native urease (left column) and urease + 2-ME (right column). Plots a and d are the raw k 3 x ( k ) data for the two samples; plots b and e are the Fourier transforms (&'-weighted, k = 3.0-13.0 A-') (- -) of the data in plots a and d, respectively, showing the windows (- --) used for the first-shell filters, which are shown as solid lines in plots c and f, respectively. The best curve fits to the first-shell filtered data are shown as the dashed lines in plots c and f and correspond to fits 1 and 6 in Table 11, respectively.
of the specific activity based on &IO and the Specific activity based on Ni content (maximum value 1.33 x 10'' IU/Ni) indicates the urease samples had ca. two Ni atoms per subunit. The XAS samples were run as frozen glasses in 50-60% glycerol. Nickel concentrations (Table I) were determined by atomic absorption spectroscopy. Urease samples exposed to the X-ray beam retained >90% of their original activity. The nickel X-ray absorption edge spectrum of native urease is compared in Figure l a with the edge spectrum of the approximately octahedral NiN6 compound [Ni(en)3]C12.2H20(en = 1 ,Zethylenediamine). The generally featureless edge shape is characteristic of pseudo-octahedral Ni(I1) geometry.* The slightly enhanced intensity of the 8332-eV 1s 3d transition in the native urease edge is indicative of a slight distortion from the ideally centrosymmetric octahedral geometry.* Curve-fitting analysis of the Ni EXAFS region (Figure 2, Table 11) confirms the presence of five or six (N,O)-containing ligands at an average Ni-(N,O) distance of 2.06 A. These results are in substantial agreement with an earlier XAS determination of the native urease Ni(I1) site structure (using lower quality EXAFS data)I0 and with magnetic4 and spectroscopic"J* evidence for a pseudooctahedral Ni(l1) site. Addition of 2-ME to native urease causes changes in the Ni X-ray absorption edge and EXAFS spectra as shown in Figures
-
Table 11. Curve-Fitting Results for the First Coordination Spheres of Jack Bean Urease and Its 2-Mercaptoethanol Complex" Ni-(N,O) NCS sample fit N R, A Aa2, A2 N R, A Pa2, A2 f f b oxidized 1 6c 2.06 -0.0013 0.02 1 2 5 2.06 -0.0026 0.020 3 5 2.06 -0.0026 1 2.10 0.6066 0.020 4 3 2.06 -0.0034 0.020 3 2.07 +0.0018 oxidized + 2-ME 5 6 2.08 -0.0012 0.034 6 5 2.07 -0.0020 1 2.29 -0.0000 0.021 7 3 2.02 -0.0060 0.024 3 2.14 -0.0065 a N is the number of scatterers per nickel; R is the nickel-scatterer distance; Au2 is a relative mean square deviation in R , ha2 = a2(sample) - u*(reference),where the reference is [Ni(en)3]C12.2H2015 at 4 K for Ni-(N,O) and [(C6H5)4P]2[Ni(SC6H5)4]16 at 4 K for Ni-S. All fits were over the range k = 4.0-12.0 A-l. Errors in R and Au2 are estimated to be f0.03 A and +0.0050/-0.0025 A2 for Ni-(N,O) and *0.02 A and +0.0019/-0.0013 A2for Ni-(S9Cl).17 bf'is a goodnessof-fit statistic normalized to the overall magnitude of the k3x(k) data:
f'=
I Z [ k 3 ( x d i )- ~ ~ d i ) ) l ~ / N l ' ' ~ (k'~)max
- (k'XX)min
Coordination numbers were not varied during optimization. (IO) Hasnain, S.S . ; Piggott, B. Biochem. Biophys. Res. Commun. 1983,112, 279-283. Alagna, L.; Hasnain, S. S.; Piggott, B.; Williams, D. J. Biochem. J . 1984, 220, 591-595. ( I I ) Blakeley, R. L.; Dixon, N . E.; Zerner, B. Biochim. Biophys. Acta 1983, 744, 219-229. (12) Clark, P. A.; Wilcox, D. E. To be submitted for publication.
1b and 2d, respectively. The slight shift of the edge to lower energy and decrease in the edge height are both consistent with an increased covalency of the Ni(I1) site expected from coordination of a sulfur-containing ligand.* The curve-fitting results (Table
58 1
Inorg. Chem. 1990, 29, 581-582 II) confirm the requirement of one (S,CI)-containing ligand (at a Ni-(S,Cl) distance of 2.29 A) in addition to approximately five (N,O)-containing ligands to simulate the first-shell Ni EXAFS of the 2-ME-bound urease derivative. Although the improvement inf' upon addition of a Ni-(S,CI) shell to the simulation for the 2-ME-bound derivative (fit 6, Table 11) is less than a factor of 2 and could result from simply the increase in the number of parameters, we have attempted other fits, the results of which suggest that the (S,CI)-containing ligand is required to fit the data. For example, providing the same additional parameters to fit the oxidized urease data does not result in improvement inf', whether this is from a (S,CI) shell (fit 3) or an additional (N,O) shell (fit 4). Another two-shell fit of the data for the 2-ME-bound derivative (fit 7) using different (N,O) shells does result inf' improvement (suggesting that two separate shells are required for this derivative but not for the oxidized derivative), but the resulting Ni-(N,O) distance for the second shell (2.14 A) is chemically unreasonable. The simplest explanation of these curve-fitting results involves the average Ni(N,O),(S,CI) coordination sphere discussed above. The Ni-(S,CI) Debye-Waller factor derived from EXAFS curve-fitting is similar to that of the [(c6H,)4P]2[Ni(sc,H,)4] model only for a coordination number of 1 (not OS), supporting the binding of one 2-ME thiolate to each nickel or one thiolate simultaneously binding both. The similar intensity of the 8332-eV peak in the native and 2-ME-bound derivatives (Figure lb) indicates the same slight distortion from octahedral symmetry, suggesting that 2-ME binding is a simple ligand-exchange reaction. The diamagnetism observed upon 2-ME treatment of urease4 thus correlates with direct binding of 2-ME to Ni(I1) through the thiolate sulfur. Square-planar or substantially tetragonally distorted ligand fields are required for mononuclear Ni(I1) compounds to adopt low-spin (diamagnetic) electronic configurations. Our edge data preclude such a large distortion from octahedral symmetry for the urease Ni(I1) ions since the transition at 8336 eV, the characteristic signature of tetragonal geometries,* is not observed. The alternative explanation for the diamagnetic ground state involves creation of strong antiferromagnetic coupling between the two high-spin (S = 1) Ni(I1) ions upon 2-ME binding. Our structural results are fully consistent with the 2-ME thiolate sulfur bridging the two Ni(I1) ions and mediating this antiferromagnetic exchange interaction.13 In such a model, the competitive nature of the 2-ME inhibition would imply a substrate-binding site involving both Ni(I1) ions. Future XAS studies will target identification of the Ni-qNi scattering expected from such an inhibitor-bridged binuclear site. Acknowledgment. Support for this research has come from the USDA (Grant 87-CRCR-1-2485) to D.E.W. and from the N S F (Grant 86-45819) to R.A.S. The XAS data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL), which is funded under Contract DE-AC03-82ER-13000, by the Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, and the National Institutes of Health, Biotechnology Resource Program, Division of Research Resources. R.A.S. is a Presidential Young Investigator.
(13) We cannot at this time rule out the possibility that one 2-ME molecule
binds to each Ni(1l) and that the strong magnetic coupling results from some indirect effect causing a large increase in the weak intrinsic magnetic interaction between the Ni(I1) ions found' in native urease. (14) Cramer, S . P.;Tench, 0.; Yocum, M.; George, G. N. Nucl. Instrum. Methods Phys. Res. 1988, A266, 586-591. (15) Curtis, N. F. J . Chem.Soc. 1961, 3147-3148. (16) Swenson, D.; Baenziger, N. C.; Coucouvanis, D. J . Am. Chem. SOC. 1978, 100. 1932-1934. ( I 7) Scott, R. A.; Schwartz, J. R.; Cramer, S . P.Biochemistry 1986, 258 5546-5555.
0020-1669/90/ 1329-0581$02.50/0
Registry No. 2-ME, 60-24-2; Ni, 7440-02-0; urease, 9002-1 3-5.
Department of Chemistry Dartmouth College Hanover, New Hampshire 03755
Patrick A. Clark Dean E. Wilcox*
Departments of Chemistry and Biochemistry and The Center for Metalloenzyme Studies University of Georgia Athens, Georgia 30602
Robert A. Scott*
Received October 5, 1988
Comparing the Acidity of Hydride and $-Dihydrogen Complexes of Transition Metals The need for the measurement of the acidity of metal hydride complexes has been stressed, and the pK, values for a number of carbonyl metal hydride complexes have been determined-I There are scattered reports that the proton of the dihydrogen ligand is acidic: for example, in [CpRu(q2-H2)(dmpe)]' (dmpe = PMezCH2CH2PMeZ),2[IrH(qz-H2)(bq)(L)2]+(bq = 7,8-benzoquinolinate; L = PPh,, PCy,)? [MH4(qz-H2)(dppe)z]' (M = Ru, Fe; dppe = PPh2CH2CH2PPh2), [FeH(q2-Hz)(dmpe)z]+,5 [Cp*Ru(C0),(q2-Hz)]+,6 and [Cp*Re(CO) (NO) (q2-Hz)]+.6The q2-dihydrogen ligand is known to be deprotonated in preference to the terminal hydride in the complex [IrH(q2-Hz)(bq)(L)2]+3 and in the mixture of complexes [CpRu(qZ-Hz)(dmpe)]+and [CpR~(H)~(dmpe)]'.~We report here a simple method for the ranking of the acidity of a range of q2-dihydrogen and dihydride compounds [CpRu(q2-Hz)(dppm)]+(dppm = PPh2CHzPPhz),7 [ C p R u H z ( d ~ ~ e ) l + , ' ~ *[ C P R U ( H ) Z ( ~ P P P ) I +( ~ P P P = PPh2CH2CH2CH2PPhz),7[ C ~ R U ( H ) ~ ( P P ~ ~and ) ~ ][MH+,~ (qZ-H2)(dppe)2]+( M = Fe, Ru, O S ) . ' ~We also describe how approximate pK, values can be obtained. The method involves the determination of the equilibrium constant Kq for the following reaction by N M R spectroscopy: M H M'H2' s MH2" M'H (1)
+
+
We choose dichloromethane as the solvent because it is noncoordinating and it dissolves the neutral and ionic metal complexes without reaction. Although CH$N is the preferred solvent for hydride pK, determinations,'v2 it displaces H2 from most dihydrogen complexes including the ones described in this work. In a typical experiment appropriate amounts of a neutral compound and an ionic complex were loaded into an N M R tube and then CDzC12 was added. After a period was waited to let the system reach equilibrium," a IH N M R spectrum was recorded; a typical example is shown in Figure 1. By measuring the intensity of the hydride resonances, one can calculate the relative (1) Moore, E. J.; Sullivan, J. M.; Norton, J. R. J . Am. Chem. SOC.1986, 108, 2257 and references cited there. (2) Chinn, M. S.;Heinekey, D. M . J . Am. Chem. SOC.1987, 109, 5865. (3) Crabtree, R. H.; Lain, M.; Bonneviot, L. J. Am. Chem. Soc. 1986,108, 4032. (4) Morris, R. H.; Sawyer, J. F.; Shiralian, M.; Zubkowski, J. D. J . Am. Chem. SOC.1985, 107, 5581. (5) Baker, M. V.; Field, L. D.; Young, D.J. J . Chem. SOC.,Chem. Commun. 1988. 546. (6) Chinn, M.S . ; Heinekey, D. M.; Payne, N. G.; Sofield, C. D. Organometallics 1989, 9, 1828. (7) Conroy-Lewis, F. M.; Simpson, S. J. J . Chem. Soc., Chem. Commun. 1987. 1675. (8) In sohion, [CpRuH2(dppe)]+ is a rapidly interconverting 1:2 mixture
of [CpRu(q2-H2)(dppe)]+and [C~Ru(H)~(dppe)]+ forms, respectively.' (9) Wilczewski, T. J. Organomet. Chem. 1989, 361, 219. (10) (a) Bautista, M. T.; Earl, K. A.; Maltby, P. A.; Morris, R. H.; Schweitzer, C. T.; Sella, A. J . Am. Chem. Soc. 1988, 110, 7031 and reference therein. (b) Cappellani, E. P.;Maltby, P.A.; Morris, R. H.; Schweitzer, C. T.; Steele, M. R. Inorg. Chem. 1989, 28, 4437. (1 I ) The equilibrium is usually reached in less than 30 min. For example, the K values obtained 30 min or 4 h after mixing the reactants did not
differ appreciably.
0 1990 American Chemical Society
